Assembly of apical actin ring and epithelial extrusion rely on myosin activity. (A and B) MCF10A cells transfected with mCherry-MRCKα 1–478 show, in correspondence with iEAAR, enrichment of phosphorylated regulatory subunit of myosin, detected with pS19-MLC2 (A) or pT18/S19 MLC2 (B) antibodies. (C) STED nanoscopy image of an iEAAR stained for F-actin and pT18/S19 MLC2. (D) Cells were transfected with mCherry-MRCKα 1–478 in the presence or absence of 100 µM blebbistatin and stained for F-actin (green) and pT18/S19 MLC2. Bars in A, B, and D, 10 µm. (E and F) Cells were transfected with mCherry alone (as negative control) or mCherry-MRCKα 1–478 in the presence of increasing concentrations of blebbistatin and evaluated for the frequency of iEAAR formation (E) and the frequency of extrusion (F). (G and H) Subconfluent MCF10A cells were cotransfected with GFP-actin and mCherry-MRCKα 1–478. Upon iEAAR assembly completion, cells were treated with 100 µM blebbistatin, and 10 min later, actin bundles (G) or the iEAAR itself (H) were bleached and monitored by time-lapse laser scanning confocal microscopy. (I) Actin bundle flow speed measured on the bleached region of cells treated or untreated with blebbistatin proves that myosin contraction is required for actin flow in bundles. Error bars represent the SD. (J and K) Fluorescence recovery of GFP-actin of iEAAR in absence (J) or presence (K) or of blebbistatin treatment. The fluorescence recovery curve of iEAAR shows a typical biphasic behavior that is abrogated by blebbistatin treatment. ***, P < 0.001.